Polyketides are one of the most important natural products used in the healthcare industry and include antibiotics, immunosuppressant's and chemotherapeutics. The multi-domain enzyme complex that is responsible for the biosynthesis of polyketides is known as the polyketide synthase (PKS). PKS utilize various polyketide chain lengths, modification reactions and cyclization reactions to promote polyketide diversity. The cyclization reactions occur in the product template (PT) domain of the fungal PKS and are an important factor in promoting diversity and bioactivity amongst polyketides. There is a fundamental gap in understanding the general mechanism of cyclization specificity between different PT domains of varying polyketide species. The long-term goal is to better understand the generic cyclization mechanism of different PKSs in order to create novel polyketide derivatives by means of protein engineering. The objective of this application is to determine the crystal structure and sequence-structure function relationship of multiple PT domains from various fungal species. Our rationale is that we can utilize structure directed mutagenesis to change the substrate specificity of PTs in an expected manner to produce novel polyketides derivatives. With relevant preliminary data acquired, we will test our hypothesis by engaging in three specific aims: (1) Determine the protein structures of PTs with varying cyclization patterns using synthetic analogues as substrates (2) conduct a series of mutations to propose a generic cyclization mechanism and synthesize new polyketides in a predictable manner (3) probe protein-protein interaction with mechanism-based cross-linkers to stabilize PT and acyl carrier protein (ACP) interactions. This approach is significant in understanding the general cyclization mechanism in different fungal PKSs. This will be an innovation in the identification and prediction of novel polyketide derivatives for the use in healthcare and other related fields, whic is relevant to the NIHs mission statement.